In a liquid chromatograph using a photodiode array (PDA) detector or similar multichannel detector, three-dimensional chromatogram data can be obtained with respect to the three dimensions of time, wavelength and absorbance by repeatedly obtaining an absorbance spectrum for an eluate from a column, with the point of injection of the sample into the mobile phase as the base point. FIGS. 13A and 13B are model diagrams showing one example of the three-dimensional chromatogram data. By extracting data obtained at a specific wavelength from the three-dimensional chromatogram data, a wavelength chromatogram showing the relationship between time and absorbance at the specific wavelength can be created. Furthermore, by extracting data obtained at a specific point in time from the three-dimensional chromatogram data, an absorbance spectrum showing the relationship between wavelength and absorbance at that point in time can be created.
A normal procedure for a quantitative analysis of a known kind of target component in the previously described type of liquid chromatograph includes the steps of obtaining a wavelength chromatogram at an absorption wavelength corresponding to the target component and calculating the quantity value by comparing the area (or height) of a peak originating from the target component on that chromatogram with a calibration curve.
There is no problem with such a quantitative determination of a target component if the aforementioned peak on the wavelength chromatogram originates from only the target component. However, a peak is not always composed of a single component (i.e. the target component); it is often the case that a peak contains an unintended impurity. To address this problem, a peak purity determination process has conventionally been performed in order to determine whether the peak in question on the chromatogram originates from only the target component or contains an impurity.
For example, Patent Document 1 discloses a technique for determining the purity of a peak on a chromatogram obtained by a liquid chromatograph using a multichannel detector. In this technique, a degree of matching P of two absorbance spectra S0(λ) and S(λ) is calculated by the following equation (1):
                    P        =                              ∑                                                            S                  0                                ⁡                                  (                  λ                  )                                            ·                              S                ⁡                                  (                  λ                  )                                                                                        ∑                                                                    S                    0                    2                                    ⁡                                      (                    λ                    )                                                  ·                                  ∑                                                            S                      2                                        ⁡                                          (                      λ                      )                                                                                                                              (        1        )            where S0(λ) is an absorbance spectrum at time T0 corresponding to the peak top of a target peak on a wavelength chromatogram and S(λ) is an absorbance spectrum at an arbitrary point in time T before or after T0. The calculated result is graphically shown in such a manner that the target peak is divided into segments along the time axis, each segment painted in a unique color corresponding to the degree of matching P with the peak top, as shown in the examples of FIGS. 14A and 14B, where green represents the degree of matching P of 1.0-0.8, yellow represents 0.8-0.6, and orange represents 0.6 or less. (It should be noted that those colors are represented by different patterns in those figures.)
If the target peak originates from only the target component, the degree of matching P is highest in the vicinity of the peak top and gradually decreases as being apart from the peak top, as shown in FIG. 14A, with the pattern of the segments being approximately symmetrical with respect to the central axis of the peak. By contrast, if an impurity peak exists before or after the peak top of the target peak (i.e. if the target peak contains an impurity), the degree of matching P decreases before or after the peak top. For example, in FIG. 14B, the degree of matching P on the right side of the peak top (in a later range of time) is lower than on the left side. Thus, it is possible to determine that an impurity is likely to be contained within a range around this point in time.
However, in the previously described conventional peak purity determination method, if an impurity peak exists at a position extremely close to the peak top of the target peak, the degree of matching P in a range close to the peak top does not noticeably decrease, so that it is in some cases impossible to correctly detect the presence of the impurity.
As described in Non-Patent Document 1, the previously described peak purity determination method requires setting a noise vector (whose components indicate, for example, the magnitude of a noise at each wavelength) as a parameter for calculating a threshold of the degree of matching P for determining whether or not an impurity peak exists. However, obtaining a noise vector requires complex calculations, such as the sequentially monitoring of the magnitude of the noise over a predetermined range of wavelengths detected by a multichannel detector as well as the calculation of the standard deviation of the temporal change in the noise within that wavelength range.
In the previously described liquid chromatograph, if there are two target components whose quantities need to be determined, and if the retention times of those two components are close to each other, the peaks originating from the target components may be insufficiently separated and overlap each other on the eventually obtained chromatogram, as shown in FIG. 15A. In a conventional process for dealing with such a situation, the two peaks with the tailing and leading portions overlapping each other are vertically divided into front and rear sections as shown in FIG. 15A, the area of each section is calculated, and the quantities of the two components X and Y are respectively computed from the calculated areas. However, the accuracy of the quantity determined in this manner cannot be high since the peak sections obtained by the vertical division do not reflect a true waveform of the elution profile of each component (i.e. the peak waveform to be obtained if the other component is not present).
Another example of the method for separating component peaks is disclosed in Patent Document 2, in which the peak separation is achieved by a computational process other than the vertical division. However, the therein described computational process is rather complex and requires a considerable amount of time. Furthermore, as shown in FIG. 15B if the peaks of the two target components are entirely superposed on each other (with one peak entirely contained in the other), the peaks cannot be separated by any of the previously mentioned techniques, and therefore, it is impossible to determine the quantity of each component.
A flow injection analysis (FIA), which does not use a column (i.e. which includes no component separation), may be used in a quantitative analysis, particularly in the case where the sample to be analyzed contains only a single component. In the FIA method, a predetermined amount of sample is injected in a mobile phase being supplied at a constant flow rate, using an injector for a liquid chromatograph or similar device. The injected sample is carried by the stream of the mobile phase and introduced into a detector. As in the case of an eluate exiting from a column, the concentration of the target component in the mobile phase changes with time, forming a roughly bell-shaped curve. If the sample thus introduced by the FIA method is detected with a multichannel detector, the obtained data will be a three-dimensional data having the three dimensions of time, wavelength and absorbance, which is substantially the same as the previously mentioned data collected by a liquid chromatograph. Accordingly, the term “three-dimensional chromatogram data” as used in the present description should be interpreted as inclusive of the three-dimensional data collected by the FIA method.